The broad long term objectives of the research to be supported by this grant are to gain an understanding at the molecular level of the mechanism of the activation and inactivation of an effector of gene transcription in response to environmental signals and of the specific conformation of the effector that allows it to act on the RNA polymerase-promoter complex. In particular we shall study the interaction between NRI, the effector of transcription of nitrogen-regulated genes of enteric bacteria, NRII, the kinase that converts NRI to the active phosphate, PII, a protein that causes NRII to remove the phosphate from NRI-phosphate and of other regulatory proteins suspected to play a role in this process. We shall attempt to identify the histidine and aspartate groups of NRII and NRI, respectively, that carry the phosphate and to discover in what manner the attachment of a phosphate group converts NRI to the activator of transcription. We shall use biochemical methods to study interaction of the highly purified proteins NRI, NRII, and PII. We shall use site directed mutagenesis to identify the phosphorylation site on NRI and use genetic recombination between glnG, the structural gene for NRI, and nifA, a gene coding for an activator with structural similarity in its central domain to NRI that appears to be active in a non-phosphorylated state, to discover the role of phosphorylation in the activation of NRI. These studies should increase our understanding of a problem of great importance for all living cells, the regulation of gene expression. The generality of the mechanisms responsible for this regulation is emphasized by the fact that the effector NRI, like effectors in the cells of higher organisms is subject to covalent modification and exerts its effect when bound to DNA at enhancers located far from the binding site of a specific RNA polymerase.